Studying the Glial Contribution to RTT Pathogenesis Using Patient-Specific iPSCs Abstract Rett syndrome (RTT) is an autism spectrum disorder (ASD) that predominantly affects females. The identification of mutations in the X-linked MECP2 (methyl-CpG binding protein 2) gene as the cause of RTT has led to the creation of mouse models for studying disease mechanism. However, mouse models have limitations in mimicking human RTT mutations and in drug screening. Induced pluripotent stem cells (iPSCs) have been generated from RTT patients as an in vitro human model to validate and extend knowledge obtained from mouse studies. Currently, all studies involving RTT iPSCs have focused on studying neuronal pathologies in the absence of astrocytes. As the other major cell type in the brain, astrocytes also express MeCP2. There is strong evidence, from work in RTT mouse models, that astrocytes play a critical role in disease progression. Thus, we hypothesize that astrocytes differentiated from mutant RTT-iPSC lines will significantly impair neuronal growth and maturation when cultured together with neurons. In the current proposal, we will focus our attention on revealing the glial contribution to RTT pathology in a neuron/astrocyte co-culture system. To minimize phenotypic variation caused by different genetic backgrounds across iPSC lines generated from different individuals, we have established several pairs of isogenic iPSC lines from the same female RTT patients (carrying either common or rare RTT mutations) skin cells that clonally express either the wild type copy or the mutant copy (but not both) of the MECP2 gene. Using these unique tools, we plan to test 1) whether mutant iPSC-derived astrocytes may impair the growth and maturation of wild type iPSC-derived neurons in the neuron/astrocyte co-culture; 2) whether wild type iPSC-derived astrocytes may rescue the growth and maturation defects of mutant type iPSC-derived neurons in the neuron/astrocyte co-culture; and 3) whether the potential astrocyte influnce is mediated by cell-cell contact or secreted molecules. Better understanding of the glial contribution to RTT pathology will not only provide insight into disease mechanisms, but also lay the groundwork for future drug screens using RTT iPSC-derived neurons and/or astrocytes. Furthermore, in light of the recent evidence that MECP2 may be altered at both the genomic level and the expression level in many autism patients, the lessons learned and the experimental approaches used in studying RTT might also benefit the general understanding of autism.